Gold Nanoparticle-Based Assay Enables Rapid Detection of Dengue Virus

A rapid colorimetric agglutination assay for the detection of Dengue virus comprises reagents that are stable at room temperature and is sensitive and simple enough for field use.

The Dengue virus infects from 50-100 million people per year world-wide, and approximately 2.5% of infected individuals die from the disease, which has no vaccine or effective antiviral therapy. The main method for preventing spread of the disease is through control of the mosquito vector.

Investigators at Notre Dame University (IN, USA) sought to develop a rapid assay to replace the laborious methods currently employed for detecting the virus in mosquito cells and cultures.

They described the development of an assay system based on gold nanoparticles (AuNPs) coated with a DNAzyme specific for the Dengue virus RNA genome. DNAzymes are DNA molecules that have the ability to perform a chemical reaction, such as catalytic action. In nature DNA is only associated only with gene replication. This is due to DNA's lacking the 2'-hydroxyl group of RNA, which diminishes its chemical reactivity and its ability to form complex tertiary structures, and that nearly all biological DNA exists in the double helix conformation in which potential catalytic sites are shielded. For these reasons, DNAzymes exist only in the laboratory.

When AuNPs coated with DNAzyme specific for Dengue virus RNA were introduced into cultures of Dengue-infected mosquito cells in the presence of magnesium and heat (37 degrees Celsius) the DNA-enzyme cleaved the viral RNA. This cleavage resulted in deshielding and aggregation of the AuNPs in the presence of NaCl, causing a visually detectable red to clear color transition that could be quantified by UV/Visible spectrophotometry at 520 nanometers wavelength. If Dengue viruses were not present in the sample, the DNAzyme-coated AuNPs remained in a dispersed state and no color loss occurred. Likewise, if any of the essential components such as magnesium or sodium were not present in the reaction mixture, no aggregation was possible. The inclusion of the detergent sodium dodecyl sulfate (SDS) in the reaction mixture permitted the detection of Dengue virus directly from cell culture supernatants without additional sample processing.

The assay was shown to be able to detect as few as 10 viruses in samples containing from 10-20 mosquitoes. Test components were stable above 30 degrees Celsius, which would allow for storage and transportation under field conditions.

First author Dr.James Carter, postdoctoral research associate at Notre Dame University, said, "Full development of our novel DNAzyme-AuNP detection method will provide a practical, rapid, and low cost alternative for the detection of Dengue virus in mosquito cells and tissues, and possibly infected patient serum, in a matter of minutes with little to no specialized training required."

Results from tests carried out with the AuNP detection system were published in the June 28, 2013, online edition of the Virology Journal.

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